Expression Analysis Of The Iridoid Synthase(LeIDS) Enzyme Gene In Lithospermum Erythrorhizon And Induction Of RNAi Transgenic Hairy Root

As an important traditional Chinese medicine,Lithospermum erythrorhizon roots have been used to treat ulcers,eczema,burns caused by fire and water and other injuries since very early,which can be attributed to the roots can synthesize naphthoquinone compound-shikonin and its derivatives(Below referred to as shikonin).Studies have shown that these metabolic products also have the function of antibacterial,anti-inflammatory,anti-tumor and other activities.In view of important medical value of shikonin,it is very important to increase the yield of shikonin.At present,although the biosynthetic pathway of shikonin has been clear studied,some of the cyclization processes involved in the late synthesis process of shikonin are still not clear.It has been shown that Iridoid synthase(CrIDS)of Catharanthus roseus is mainly involved in the Iridoid cycloterpene synthesis by nepetalactone.To investigate whether its homologous gene plays a key role in the regulation and synthesis of shikonin and its derivatives or other important secondary metabolites in Lithospermum erythrorhizon,in this study,CrIDS homologous gene LeIDS was obtained based on our previous transcriptom and genome data of Lithospermum erythrorhizon,its expression pattern was analyzed,and its RNA interference vector was constructed,and then the transgenic hairy root was obtained.These results play a foundation for the subsequent functional research of this gene.The results are as follows:The open reading frame(ORF)of LeIDS was 1170 bp,encoding 389 amino acids.Phylogenetic tree analysis showed that this protein had the highest homology with 1:3-oxo-delta(4,5)-steroid 5-beta-reductase-like in Coffee arabica,and clustered in one branch;it also show high similarity with CrIDS.Gene expression analysis results showed that LeIDS was highly expressed in the root(especially the periderm)of Lithospermum erythrorhizon and in the M9 shikonin production medium.In order to study the function of LeIDS by using the RNA interference strategy,the interference vector was constructed in this study and this vector would be transformed into agrobacterium rhizome ATCC15834.Then,two transgenic hairy roots of pBI 121-eGFP empty plasmid(EV)and interfere vector pBI 121-LeIDS-RNAi(Ii)and wild hairy root(WT)without exogenous vector were obtained by leaf explant induction.Three kinds of hairy roots were identified by fluorescence.WT hairy roots did not show any fluorescence,while EV and Ii transgenic hairy roots all contained fluorescence,which reveals that the LeIDS target gene has been successfully transferred into the hairy root cells.The results provided experimental materials for the study of LeIDS gene in regulating the biosynthesis of shikonin and its derivatives,or other important metabolites in Lithospermum erythrorhizon.